The molecular mechanisms responsible for the accumulation and restriction of chemoreceptors beneath presynaptic nerve terminals are poorly understood despite intense research on the subject in the last 15 years. Our goal is to better understand this phenomena by concentrating on defining the function of several proteins implicated in the clustering process. Experiments on cultured vertebrate muscle cells are designed to elucidate the role in this process of an Mr 43000 protein (43K) that is closely associated with acetylcholine receptors (AChRs) both in Torpedo electric organ and vertebrate skeletal muscle preparations. Quantitative fluorescence microscopy will be used to define the relative stochiometery of AChRs to 43K in different membrane regions. This will be followed by experiments on living cultured muscle cells designed to perturb the interaction of 43K with the AChR. Furthermore, we will examine the role of additional receptor associated peripheral proteins that were initially identified in the Torpedo preparations, but have recently been shown to be present in skeletal muscle cells. Finally, the functional studies of live, cultured vertebrate muscle cells will be correlated with immunoelectron microscopy experiments on muscle cells designed to precisely localize the different cluster associated proteins and cytoskeletal elements to which they potentially form linkages.